We have applied genetic engineering and gene transfer techniques to analyze cancer development. By DNA-mediated gene transfer experiments, we demonstrated the presence of transforming genes in human cell lines established from neoplastic tissues, as well as human primary tumors. The oncogenes present in carcinomas of the colon, lung, gall bladder, pancreas, urinary bladder, as well as rhabdomyosarcomas, were molecularly characterized and found to be homologous to the Kirsten-MSV transforming gene. The transforming gene present in a bladder carcinoma cell line, T24, was molecularly cloned and shown to be a mutated allele of the human c-has/bas-l protooncogene, the human genetic sequences related to the BALB-MSV transforming gene. A single base pair change was found to be directly responsible for the neoplastic properties of the oncogene isolated from the T24 cell line. Recently it was reported that the c-has/bas-l protooncogene assumed transforming activity when under the transcriptional control of viral LTR, which enhances its expression levels. We planned transfection experiments to learn whether the c-has/bas-l human protooncogene could by itself cause transformation. NIH/3T3 cells were transfected with large amounts of the c-has/bas-l protooncogene to provide recipient cells with multiple (greater than 40) copies of this protooncogene integrated into their genomes; all were characterized as transformed. However, DNA from the NIH/3T3 transformants failed to transfer the transformant phenotype in successive cycles of transfections, indicating multiple copies of the c-has/bas-l protooncogene are necessary for transformation. During characterization of the NIH/3T3 transformants containing many copies of the c-has/bas-l protooncogene, we isolated an in vitro activated oncogene in which the acquired transforming activity was switched on by a single point mutation located in the same position as that found in the T24 oncogene.